This grant supports our X-ray crystallographic studies of RNA polymerase II (pol II) and associated proteins. The work began with the crystal structure determination of pol II alone at 2.8 and of an actively transcribing complex at 3.3 . There followed the structures of transcribing complexes in both pre- and post-translocation states; structures in the post- translocation state with nucleotides bound showing the basis for specificity (trigger loop hypothesis); the structure of pol II in a backtracked state important for proofreading; the structure of an initiating complex; the structure of a cocrystal of pol II with general transcription factor TFIIB; and the structure of Mediator Head module. Specific aims for the most recent project period were 1) extension of resolution of transcribing complex structure, 2) determination of pre-initiation complex (PIC) structure, and 3) determination of Mediator structure, especially the structure of a Mediator Head module-CTD complex, and extension of structural analysis to the entire Mediator. These aims were achieved, with the completion of four very long-term projects and publication of the results: the cryo-EM structure of a 33-protein, 1.5 megaDalton PIC at 6-11 resolution; the crystal structure of a Mediator Head module-CTD complex; the architecture of the entire Mediator by Integration Modeling of data from crystallography, cryo-EM, chemical cross-linking, and other sources; and the cryo-EM structure of a 52-protein, 2.5 megaDalton Mediator-PIC complex at 16 resolution. The goal of this project is to elucidate the mechanism of transcriptional regulation. Specific aims for the next project period are as follows: 1. To extend the resolution of cryo-EM of a 52-protein, 2.5 megaDalton Mediator-PIC complex by data collection at 300 kV on a direct electron detector, and thereby to address hypotheses concerning the path of the pol II CTD and Mediator-activator interaction. 2. To test a hypothesis for the basis of the CTD cycle, which underlies transcriptional regulation. We have proposed that a 79-amino acid linker preceding the 27 heptad repeats of the CTD is the key to the CTD cycle. We are constructing a series of pol II mutants, varying in the length and sequence of the linker. 3. To elucidate the pol II transcription mechanism by time-resolved serial femtosecond crystallography. At present, we have structures of transcribing complexes before and after nucleotide addition. The proposed experiments will fill the gap between, revealing transcription with 10 ms resolution, well within the timescale of the process.